In previous studies we showed that (a) lactose synthesis and secretion were inhibited by microfilament- and microtubule-altering drugs, respectively; (b) microtubule formation is accelerated 1 week prepartum in guinea pig mammary gland, peaks with peak lactation, and reverses with weaning; (c) the increase is restricted to the apical portions of the alveolar cells in immunofluorescence studies; (d) isolated alveolar cells have twice the tubulin and twice the perentage polymerized as other cell types; (e) ovariectomized pregnant rats undergo a 3-fold increase in mammary tubulin polymerization accompanying induced lactogenesis; which (f) is blocked by progesterone but not estrogen. The purpose of the proposed project is to examine further the hormonal control of microtubule formation and function associated with lactation and to elucidate mechanisms for their regulation. We will carry this out both in vivo using ablation experiments and in vitro with short-term slice incubations, floating collagen gel cultures, and cultured explants of mammary cells and tissues. Late-pregnant rats will undergo bilateral ovariectomies under ether anesthesia and tissues collected 6,12,18,24,36 and 48 hr. Groups will include OVX, sham, and OVX plus hormone manipulations. Free and polymerized tubulin are measured using the 3H-colchicine binding assay which we adapted for mammary gland during the previous project period. Lactose will be monitored at the same time periods as an indicator of lactogenesis. We will also attempt to study the effects of increased prolactin levels using autotransplants and decreased levels using a prolactin secretion inhibitor. Slice incubations will be used to elucidate the effects of secretagogues (e.g. cyclic nucleotides) on microtubule formation and cultured cells and explants will be utilized to examine this process during hormonally-induced lactogenesis in vitro. With each of these models, one- and two-dimensional gel eletrophoresis will be carried out to determine if changes in polymerization are accompanied by alterations in tubulin or its associated proteins. Electron microscopic analysis will also be used to follow microtubule alterations throughout. These studies should elucidate not only the role of microtubules in mammary gland secretion but hormonal regulation of the cytoskeleton in lactogenesis and lactation.